We are studying the pathogenesis of viral hepatitis and the molecular basis for virulence and attenuation of these important pathogens. In collaborative studies with Dr. Frank Chisari (Scripps Institute) we studied in chimpanzees the mechanism by which the host clears a hepatitis B virus infection and the relationship of these mechanisms to clinical disease. We demonstrated that the clearance of the template for HBV synthesis, covalently closed circular HBV DNA, is eliminated from hepatocytes by non-cytolytic mechanisms mediated principally by interferon gamma in the liver. Elimination of residual hepatocytes containing HBV antigens is a later event that is mediated by cytolytic CD8 positive T cells and is temporally related to the hepatitis phase of the infection. The spectrum of virus-induced and immune response-related genes involved in acute hepatitis B were further studied by microarray analysis of intrahepatic messenger RNAs up-regulated and down-regulated during the course of hepatitis B infections in chimpanzees. Surprisingly, we could not detect evidence of an innate immune response to infection, suggesting that HBV can subvert the host immune response, but we did detect a strong adaptive immune response during the clearance phase of infection; this correlated with the inhibition of viral replication and removal of infected cells described above. Interestingly, in additional studies of chronic HBV infection in chimpanzees, we found an innate immune response that was missing in acute, self-limiting infections of HBV. This occurred at a time of liver damage, suggesting that viral and cellular products released from dead and dying hepatocytes could trigger other innate host defenses, such as TLR-3. However, the up-regulated innate immune responses were weak and insufficient to affect virus replication. The genetic heterogeneity of hepatitis C virus is believed to play an important role in its pathogenicity. We have previously examined this relationship by determining the genetic heterogeneity of HCV isolates that were recovered from patients who were infected following transfusion in order to study the early phase of infection and from patients undergoing interferon therapy in order to study changes during the later phase of chronic infection (these may be important in understanding late sequelae, such as liver cancer). Distinctive patterns of dynamic change in the sequence of viral clones during the first several weeks of infection were observed and these correlated with the outcome of infection. Similarly, the pattern of dynamic changes in sequence during interferon therapy was predictive of the outcome. These findings may be useful in predicting the outcome of therapy with interferon early in the course of treatment. Although considerable information has been gained from these longitudinal studies of patients, it is difficult to study the mechanisms of pathogenesis in such systems. Chimpanzees, which are the only animals other than man that are susceptible to infection with HCV, provide an experimental model for studying the interactions of the host and the virus in the pathogenesis of hepatitis C. Recent studies in chimpanzees suggest that even late sequelae like liver cancer can be studied in the chimpanzee. Collaborative studies with Frank Chisari have demonstrated that, as in hepatitis B virus infections, in hepatitis C virus infections the cellular immune response plays an important role in noncytolytic down-regulation of viral replication and cytolytic removal of residual infected cells. These two mechanisms are sequential and overlapping and the former appears to be mediated by interferon gamma and the latter by CD8 positive cells and, perhaps by interferon gamma through its proinflammatory activity. These studies have also revealed that type 1 interferon (interferon alpha/beta)-activated antiviral proteins are expressed in response to the viral infections, but that HCV is resistant to the antiviral activity of this innate immune response. Microarray studies of the host immune responses to viral hepatitis and how the hepatitis viruses attempt to circumvent the responses, are yielding important information on pathogenesis of these diseases, and the studies are being extended to the other hepatitis viruses in order to delineate the comparative pathogenesis of these agents in a single host, the chimpanzee, which is the only non-human host that is susceptible to all human hepatitis viruses. In 2010, we completed a microarray analysis of HCV and HEV infections of multiple chimpanzees, comparing the sequence of human and chimpanzee genomes for suitability in interpreting Affymetrix microarray data obtained from serial clinical samples of chimpanzees infected with one or the other of the two viruses. We found that the human genome sequence was more sensitive and specific for identifying up-regulated and down-regulated genes, probably because it is better curated than the more recently sequenced chimpanzee genome. We also compared two analytic methods, a method utilizing Affymetrix probe sets and correlation coefficients with one utilizing individual perfectly matched probes and t-test analysis. Both analyses demonstrated similarities and differences in the host immune response to these two RNA viruses. This information will be useful for a better understanding of the pathogenesis of viral hepatitis. Currently, we are also studying the pathogenesis of HDV infections by microarray analysis and have identified both innate and adaptive immune responses to such infections. Immune responses to HDV infection were similar to those we observed during HCV infection: both a strong innate and a strong adaptive immune response. The innate immune response was sufficient to down-regulate HBV replication in HBV chronically infected chimpanzees that were superinfected with HDV. Thus, HBV, although unable to trigger an innate immune response, is highly sensitive to such a response when provided by another virus. In 2011 we reproduced in cell culture the quasispecies evolution of HCV that had first been observed in vivo in chimpanzees that had been administered antibody to HCV. The dynamics of quasispecies change over time were remarkably similar in vivo and in vitro. In other collaborative studies, we measured changes in the HCV receptors scavenger receptor B1 (SRB1), claudin-1 and occludin after liver transplantation and the influence of such changes on early viral kinetics. There was a significant correlation between the amount of SRB1 in the liver at the time of reperfusion of the transplanted liver and the decay of the serum HCV-RNA titer, suggesting that a greater distribution of the receptor led to more rapid uptake of the virus by the liver. Similarly, higher levels of claudin-1 and occludin in the liver correlated with more rapid recurrence of HCV in the transplanted liver, additional evidence of their importance in the uptake and replication of HCV in hepatocytes. In 2012 we mapped mutations in HCV genotype 2a and 2b viruses that permitted the viruses to replicate robustly in cell culture, thereby extending the list of viruses and providing a systematic approach for adapting additional HCV strains to in vitro culture. In addition, we studied the host response to HCV infection in chimpanzees and demonstrated the dominant but transitory role of CD8+ T cells in early viral evolution.